Project Summary/Abstract Strategies for a functional cure with use of non-neutralizing antibodies (nnAbs) that eliminate HIV-infected cells through Fc receptor (FcR) effector function including antibody dependent cellular cytoxicity (ADCC) remain a significant yet largely unexploited avenue of research.This project is designed to test the hypothesis that conjugates of CD4-mimetic miniprotein M48U1 and nnAbs specific for epitopes in the first and second constant (C1-C2) region of HIV-1 Env (A32-region or Cluster A epitopes) will develop into therapeutic antibodies (tAbs) capable of efficient reduction of the infected cell reservoir in infected individuals on ART thus develop into agents capable of a functional cure exclusively through FcR-effector mechanisms. This hypothesis will be tested using set of anti-Cluster A nnAbs which recognize highly conserved and functionally critical regions of gp120 which provide breadth of killing while minimizing the possibility of epitope escape. We have characterized these epitope targets at atomic and mapped them to the discontinuous sites involving highly conserved residues of the g120 inner domain. These regions in unliganded Env trimers are important for trimer stability and are inaccessible for antibody recognition until interactions of the Env spike with the cellular CD4 receptor. Our recent data indicate that exposure of these targets on the infected cell is limited by the low levels of surface CD4 that could effectively trigger Env trimers emerging on the cell surface. This represents a highly sophisticated mechanism put in place by HIV to prevent antibody-mediated clearance of virally infected cells. We were able to overcome the unfavorable exposure of these A32-region epitopes on infected cells with a CD4-mimetic molecule, the miniprotein M48U1 which binds within the CD4 binding site of Env trimers and triggers them to assume the CD4-bound conformation required to expose the A32-region epitopes on the surface of infected cells in the sera of HIV-1 infected individuals. Accordingly, by conjugating the M48U1 with the appropriate linker to the anti- Cluster A nnAbs we aim to develop new tAbs capable of sensitizing HIV-1-infected cells to ADCC-mediated killing. This effect will result from the cooperative action of the two moieties of conjugate. The M48U1 moiety will expose the A32-region within the Env trimer which will be subsequently recognized by the A32-region nnAb arm. Aim 1 is designed to develop mAb-M48U1 conjugates using lead mAbs of the A32-region region and evaluate their ability to recognize envelope epitopes and eliminate HIV-1-infected cells through ADCC responses in in vitro and ex-vivo experiments. Aim 2 will evaluate the ability of mAb-M48U1 conjugate to reduce the size of the latent HIV reservoir in ex-vivo ?shock and kill? experiments using primary CD4+ T cells from ART-treated and untreated individuals. Pursuing our goal with A32-region conjugates, we expect to diminish the unfavorable impact of HIV-1 Nef- and Vpu-mediated CD4 down regulation that leads to reduction of CD4-inducible ADCC targets on infected cells and fully utilize the potential of nnAbs to kill the HIV-1 infected cells through ADCC. If successful, this project will add new weapons to the arsenal being built to achieve a functional cure in HIV-1- infected individuals.